1. Field of the Invention
The present invention relates to a rapid test method and assay for determining the presence of antibodies in a patient to disease-related antigens, e.g., antibodies to Mycobacterium tuberculosis, and other mycobacterial antigens.
2. Description of the Related Art
Antibodies are naturally produced biomolecules that react specifically with usually foreign biomolecules called antigens. Disease-related microbial infections, e.g., Mycobacterium tuberculosis, which causes tuberculosis (TB), are usually characterized by the production of antibodies to the specific disease-related antigens. Antibodies are also produced with other diseases and afflictions, e.g., autoimmune diseases, wherein there is often a destructive antibody response to the host. In the case of autoimmune diseases, the host supplies the disease-related antigen, a host tissue. In this case, the corresponding host antibody synthesis may have been initiated by either a foreign substance or by a host tissue not normally encountered by the host's immune system. Subsequent antibody production may proceed in the absence of the foreign substance, due to similar structural nature of the host tissue. Herein the term “disease-related antigen” includes microbial antigens and antigens associated with the host antibody response in autoimmune diseases.
Infectious diseases are widespread in the world. Military personnel, including Navy and Marine personnel, are at particular risk because of their global deployment. According to the World Health Organization, the infectious disease tuberculosis caused by Mycobacterium tuberculosis (MTB) kills about three million people every year.
There are several conventional tests for MTB, however these conventional tests all have disadvantages.
One conventional test is a purified protein derivative (PPD) skin test. In this test, a PPD is injected subcutaneously into a patient, and after 2 to 3 days, the site of injection is observed to determine whether there is hardening and/or discoloration of the patient's skin. This test is believed to be only about 50-75% accurate. In persons that have been immunized with Mycobacterium bovis BCG, there are a high percentage of false positives. Another disadvantage of the PPD skin test is that it requires a needle injection, and patient compliance is low in coming back for observation by trained medical personnel for hardening and/or discoloration. This test is typically used as a screening test for people who do not have symptoms of tuberculosis. The specificity of purified protein derivative (PPD) skin test for the detection of tuberculosis infection is compromised by exposure to environmental mycobacteria. Wilkinson R. J., Haslov, K., Rappuoli, R., Giovannoni, F., Narayanan, P. R., Desai, C. R., Vordermeier, H. M., Paulsen, J., Pasvol, G., Ivanyi, J., and Singh, M. Evaluation of the recombinant 38-kilodalton antigen of Mycobacterium tuberculosis as a potential immunodiagnostic reagent. J. Clin. Microbiol. 35(3): 553-7. March 1997.
Another conventional test is a culture test. However, this test takes up to 6 to 8 weeks since that is how long it takes for the MTB organism to grow to an amount that is visibly detectable. Another disadvantage of the culture test is that typically only about half of all samples wherein the Mycobacterium tuberculosis is present actually grow to the point where it is detectable. Thus, this method has about 50% false negatives.
Another conventional test is a chest x-ray. However, this method does not work if the person undergoing the chest x-ray has been recently infected with Mycobacterium tuberculosis. Another disadvantage is that the chest x-ray can be mis-read. Other disadvantages are the cost of the x-ray and need to subject a person to x-rays.
Another conventional test is an acid fast staining test. This method detects the most infectious patients but not those with extrapulmonary disease. In this method, patient sputum (i.e., coughed up phlegm) is smeared onto a glass slide, and a microscopic chemical stain for acid fast bacilli (AFB) is performed using the Ziehl Nielson method. The sample is then viewed under a microscope for the presence of the red staining acid-fast mycobacterium organisms. This test requires hours to complete and a high level of expertise of the microbiologist running the test. Only after sputum tests on three separate occasions have come up negative is the patient considered negative for AFB. Examination of three sputum samples detects up to 87% of pulmonary tuberculosis patients, but a considerably lower percentage of patients (43%) with extrapulmonary disease.
Another test is a MycoDot test. The MycoDot test detects anti-lipoarabinomannan IgG antibodies in serum/whole blood to diagnose cases of active TB. The MycoDot test employs the lipoarabinomannan (LAM) antigen bound to plastic combs. During incubation of the comb in the diluted serum sample for 6 minutes, the free anti-LAM antibodies, if present in the serum/blood of the individual, will bind to the antigen on the comb. Next, the excess antibodies that might be loosely attached to the comb are washed off. During incubation of the comb in a colloidal gold signal reagent for 10 minutes, the bound anti-LAM antibodies on the comb, if present, then bind to the protein ligand attached to the gold particles, providing a pink colored aggregate at the antigen spot on the comb. Next, any excess signal reagent is washed off the comb. The presence of a pink colored spot on the comb, generated as a result of the steps described above, is indicative of a positive reaction due to active TB. However, the test requires either serum or blood and takes at least 20 minutes to perform. It is also not a portable test and requires a laboratory facility to perform.
Several mycobacterial antigens are presently being exploited in the development of improved vaccines and serodiagnostic reagents. A 38-kDa antigen of Mycobacterium tuberculosis was evaluated as a potential immunodiagnostic reagent. Wilkinson R. J., Haslov, K., Rappuoli, R., Giovannoni, F., Narayanan. P. R., Desai, C. R., Vordermeier, H. M., Paulsen, J., Pasvol, G., Ivanyi, J., and Singh, M. Evaluation of the recombinant 38-kilodalton antigen of Mycobacterium tuberculosis as a potential immunodiagnostic reagent. J. Clin. Microbiol. 35(3): 553-7. March 1997. More specifically, serological tests pursuant to this method found a 72.6% sensitivity of the test in comparison with that of culture, and the specificity was reported as 94.9%.
Young et al. (3) reported that the 38 kDa antigen carries two non-overlapping epitopes, recognized by monoclonal antibodies TB71 and TB72, respectively, that are expressed substantially more strongly by MTB than by Mycobacterium bovis. Antibodies were detected in sera from tuberculosis patients estimated by competition binding against TB72 antibody using a 38-kDa-antigen-coated microtiter plates. Their results suggest the immunodominance of the species-specific B cell and cross-reactive T-cell stimulatory epitopes.
Singh et al. (4) also conclude the 38-kDa protein (Ag38) is an immunodominant antigen of potential utility for diagnosis and vaccine development. The recombinant antigen that they produced could not be distinguished antigenically from the native protein of MTB.
A screening test was described by Rosales-Borjas (5) for the diagnosis of tuberculosis (TB) by immunodot (IDt) using the 38-kDa glycoprotein antigen, which has shown great specificity in their previous serologic analyses. The serologic test was used to examine 28 sera from patients with lung tuberculosis. Of these, 85% were positive by micro-ELISA and by the IDt test described. Control sera from healthy subjects (n=20) gave negative results for ELISA and for IDt, which indicated that their screening test was highly specific. However, the sample application required a 30-minute soaking procedure followed by drying and two additional 10 minute incubation procedures plus numerous washing procedures. It also requires a laboratory facility and skilled personnel to perform.
Bassey et al. (6) provide evidence that a combination of MTB antigens may be a useful basis for developing a diagnostic antibody test.
Kadival et al. stated that the 38-kDa antigen appears to be conserved (7) and is only found in MTB and M. bovis BCG. They also observed the 38-kDa antigen may contain a specific epitope detected by serology, and also contains epitopes that are cross-reactive for cellular immunity. Unlike the 38 kDa antigen, cord factor (trehalose-6,6′-dimycolate)(TDM) has been used as a somewhat less specific antigenic marker for several mycobacteria (2, 7-10). Maekura et al. and Fujiwara et al. (2 and 8) describe ELISA procedures with TDM that are useful for the serodiagnosis of tuberculosis.
Patients with active pulmonary tuberculosis showed significantly higher titers of IgG antibodies against cord factor (TDM) than did other groups (p<0.001)(2). The ELISA had a sensitivity of 81% and a specificity of 96%. From these results, it was concluded that the detection of IgG antibodies against TDM is useful for serodiagnosis of active pulmonary tuberculosis. It also indicated that the anticord factor antibody titers decline to the normal level as a result of antituberculosis chemotherapy making TDM a possible prognostic marker.
He et al. (9) also developed an ELISA using TDM and found the sensitivity and specificity to be 83.8% and 100%, respectively. They concluded that ELISA with TDM as the antigen is simple, reproducible, and useful for the rapid serodiagnosis of general mycobacterial infections including tuberculosis.
Enomoto et al. (10) state that Mycobacterium avium-intracellulare complex (MAC) is one of the most important opportunistic pathogens, particularly in patients with acquired immunodeficiency syndrome (AIDS). Their findings suggest that ELISA using TDM is useful for rapid serodiagnosis of MAC infection.
Chang et al. (11) have expressed and purified the 16-kDa antigen from MTB, and characterize it as an immunodominant antigen with serodiagnostic value. Cunningham et al. (12) feel that the 16-kDa protein may be a marker for the dormant (latent) state of TB.
U.S. Pat. No. 4,458,014 discloses a SEROLOGICAL METHOD FOR THE IDENTIFICATION OF MICROORGANISMS for the identification of diseases of the mouth. U.S. Pat. No. 4,866,167 discloses a DETECTION OF PATIENT ORAL CELLS BY NUCLEIC ACID HYBRIDIZATION to detect oral bacterial species. The methods of both of these patents are technically complex, time consuming and are not rapid.
U.S. Pat. No. 4,639,419 discloses an IMMUNOLOGICAL COLOR CHANGE TEST INVOLVING TWO DIFFERENTLY COLORED REAGENT SPOTS. This patent discloses an agglutination reaction directed toward identifying antigenic material wherein a colored substrate and colored reagent combine, in positive reactions, to give the appearance of a third color.
There are several screening tests for tuberculosis. The Mantoux test uses tuberculin purified protein derivative (PPD) which is injected intracutaneously (e.g., Tubersol®, Connaught Laboratories Limited, Willowdale, Ontario, Canada). A delayed hypersensitivity reaction develops in individuals having previous exposure to Mycobacterium tuberculosis. The injection site is normally read within 48 to 72 hours after intracutaneous injection of the antigen; a palpable induration measuring 10 mm in diameter or more is considered a positive reaction. This procedure is accepted as an aid in the diagnosis of tuberculosis infection.
The Heaf test uses a multiple puncture disk which introduces needles through concentrated Old Tuberculin applied to the skin. The tine test uses tuberculin adhering to metal tines which is then inoculated by simple pressure into the skin. The Heaf and tine tests are acceptable for screening but should be confirmed by the Mantoux test. Antigenic material can also be applied by scratch, i.e., Pirquet's test. Similar to the Mantoux test, these invasive tests generally require 48 to 72 hours after inoculation before results can be determined. The Bacillus of Calmette and Guerin (BCG) is a live, attenuated strain of Mycobacterium bovis which has been used with varying success as a vaccine against tuberculosis in countries where the prevalence of tuberculosis is high. BCG causes tuberculin conversion to positive; it has also been used to stimulate the immune system against a variety of medical conditions. Both PPD and BCG can serve as suitable antigens to detect general mycobacterial infections with this method.
Other antigens have been described. Maes has described A60-ANTIGEN FROM MYCOBACTERIA AND USE THEREOF AS TUBERCULIN VACCINE in U.S. Pat. No. 4,965,192. This patent describes the A60-antigen as being effective for detecting prior exposure of an individual to Mycobacterial infections through the use of a cutaneous test. This patent is similar to other invasive inoculation tests mentioned earlier except that a new antigen is used and 24 to 48 hours are required to observe the responses at the test site.
To summarize, the conventional serological methods do not provide a non-invasive, rapid diagnostic test that measures exposure to mycobacteria, including MTB. A simple, rapid and non-invasive screening method would be especially useful in evaluating MTB exposure, such as for sailors and marines returning from endemic areas.